In microscopy referencing the image plane of an objective lens to a desired plane with respect to the sample can be difficult. This difficulty may be due to variations over time in the distance between the objective lens and the sample holder. Causes of such variations include changes to the sample stage, variations in sample slides, changes in position of the objective lens, thermo mechanical fluctuations, and any combinations thereof. Knowing the position of the objective lens with respect to a sample holder is valuable for focusing the objective lens.
Typical focusing methods are derived from image analysis through human or software interpretation. In traditional light microscopy, the user manually adjusts the mechanical focus mechanism while simultaneously looking into an eyepiece for an image to appear. As this image comes into focus, the user fine tunes the focus to obtain the clearest image. This is the most rudimentary form of feedback. As camera sensitivity has improved, imaging lower light levels has become possible, for example in fluorescence microscopy single fluorophore imaging is now attainable.
With these high sensitivity imaging systems, there are two basic methods of focusing. A first method requires a user to adjust the focus manually while viewing the image on a video monitor. Although this method works, it requires skill and experience, particularly when initially finding the focus point. In high magnification systems the depth of field is small so that maintaining the focus is difficult given that any “X-Y” movement of the stage will usually require refocusing given that there will be slight movement of the sample in the “Z” axis. In addition, stage drift due to thermal fluctuations is another common source of mechanical drift.
Another method requires a user to employ digital image based software to drive an auto focusing mechanism. Image analysis is used to determine focus by measuring contrast of an acquired digital image. This requires that the system have a motorized focus driven from a computer that simultaneously analyzes the image. To do this, an image from the camera is captured by the computer and analyzed. This provides a control signal that drives the motorized focusing system in a repeating cycle until the proper focus is obtained. This works well to initially get a sample focused. At this point any refocusing must be initiated by a command. While it is refocusing, the camera is dedicated to the focusing process, itself. Unfortunately, this process is slow and precludes image acquisition during the automated focusing.
A reliable way to reference an objective lens image plane to a sample without using an image of the sample in the image plane is needed.